Name: analysis of snare-mediated membrane fusion using an enzymatic cell fusion assay
Uploaded: 2012-10-19T13:00:00
Description: Watch this Scientific Journal Video about Analysis of SNARE-mediated Membrane Fusion Using an Enzymatic Cell Fusion Assay at JoVE.com

Dette arbejde understøttes af startup midler fra University of Louisville og CA135123 fra National Institutes of Health (til CH).

1. Celledyrkning og transfektion <ol><li> COS-7-celler dyrkes i Dulbeccos modificerede Eagles medium (DMEM) suppleret med 4,5 g / l glucose og 10% føtalt bovint serum (FBS). </li><li> Plasmid transfektion udført med lipofectamin ifølge producentens instruktioner (Invitrogen). </li></ol> 2. Fluorescence mikroskopisk analyse af Cell Surface SNARE Expression <ol><li> Dagen før transfektion, 3 × 10 4 COS-7-celler podes på sterile 12-mm dækglas (Fisher Scientific) i 24-brønds pl...

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Den oprindelige cellefusion assay 6 bestemmer SNARE-medierede celle fusionsbegivenheder ved fluorescensmikroskopi. Her beskriver vi en innovativ assay, der kvantificerer SNARE-medierede celle fusionsbegivenheder med aktiveret ekspression af β-galactosidase og spektrometrisk måling. Anvendelse af dette assay, vi rutinemæssigt analyseres 15-20 v-og t-SNARE kombinationer i et enkelt eksperiment. Ved anvendelse af flowcytometri til måling SNARE ekspression på celleoverfladen, vi titreres ekspressionsniveauer...

<table border="1"><tbody><tr><td> Navn reagens og udstyr </td><td> Firma </td><td> Katalog nummer </td></tr><tr><td> DMEM </td><td> Invitrogen </td><td> 12800-017 </td></tr><tr><td> COS-7 celler </td><td> ATCC </td><td> CRL 1651 </td></tr><tr><td> tunicamycin </td><td> Sigma-Aldrich </td><td> T7765-5mg </td></tr><tr><td> pTet-Off </td><td> Clontech </td><td> K1620-A </td></tr><tr><td> pBI-G </td><td> Clontech </td><td> 6150-1 </td></tr><tr><td> lipofectamin </td><td> Invitrogen </td><td> 18324-012 </td></tr><tr><td> Enzymfri celledissocieringsopløsning </td><td> Invitrogen </td><td> 13151-014 </td></tr><tr><td> Kanin-anti-tet-repressoren polyklonalt antistof </td><td> Millipore </td><td> AB3541 </td></tr><tr><td> FITC-konjugeret æsel-anti-muse-IgG (H + L) </td><td> Jackson ImmunoResearch </td><td> 715-095-150 </td></tr><tr><td> Laserscanning konfokal mikroskop </td><td> Olympus </td><td> FV1000 </td></tr><tr><td> FACSCalibur flowcytometer </td><td> BD Biosciences </td><td></td></tr><tr><td> β-galactosidase Enzyme Assay System med Reporter Lysis Buffer </td><td> Promega </td><td> E2000 </td></tr><tr><td> Model 100-40 UV-VIS spektrofotometer </td><td> Hitachi </td><td> C740843 </td></tr></tbody></table>

analysis of snare-mediated membrane fusion using an enzymatic cell fusion assay

De interaktioner mellem SNARE (s oluble N-ethylmaleimid-følsom faktor en ttachment protein re ceptor) proteiner på vesikler (v-Snares) og på målgrupperne membraner (t-Snares) katalyserer intracellulære vesikelfusion 1-4. Rekonstitutionsanvisninger assays er afgørende for at dissekere mekanismen og reguleringen af SNARE-medieret membranfusion 5. I en cellefusion assay 6,7, er SNARE proteiner udtrykt ektopisk på celleoverfladen. Disse &quot;vendt&quot; SNARE proteiner drive celle-celle fusion, hvilket viser, at snarer er tilstrækkelige til at smelte cellemembraner. Fordi cellefusion assay er baseret på mikroskopisk undersøgelse, er mindre effektiv, når anvendt til at analysere multiple v-og t-SNARE interaktioner kvantitativt. Her beskriver vi en ny assay 8, der kvantificerer SNARE-medierede celle fusionsbegivenheder med aktiveret ekspression af β-galactosidase. Tokomponenter i Tet-Off genekspressionssystem 9 anvendes som en tæller systemet: tetracyclin-reguleret transaktivator (tTA), og et reporterplasmid, der koder for LacZ-genet under kontrol af tetracyclin-responselement (TRE-LacZ). Vi transficere tTA i COS-7-celler, som udtrykker vippet v-SNARE proteiner på celleoverfladen (V-celler) og transficere TRE-LacZ i COS-7-celler, som udtrykker vippet t-SNARE proteiner på celleoverfladen (T-celler) . SNARE-afhængig fusion af V-og T-celler resulterer i binding af tTA til TRE, den transkriptionelle aktivering af LacZ og ekspression af β-galactosidase. Aktiviteten af ​​β-galactosidase kvantificeres ved hjælp af en kolorimetrisk metode ved absorbans ved 420 nm. De vesikel-associerede membranproteiner (overlæder) er V-Snares, der bor i forskellige post-Golgi vesikulær rum 10-15. Ved at udtrykke Vamps 1, 3, 4, 5, 7 og 8 på samme niveau, sammenligne vi deres membran fusion, som anvender den enzymatiske cellefusion assay. Ud fra spektrometrisk måling, giver dette assay en kvantitativ fremgangsmåde til analyse SNARE-medieret membranfusion og for højt gennemløb undersøgelser.

At udvikle en kvantitativ cellefusion assay udnytter vi den stærke transkriptionel aktivering ved binding af tTA til TRE. I fravær af tTA, er transskription af lacZ-genet i TRE-LacZ tavse. Når tTA er til stede, bindes den til TRE og aktiverer transkriptionen af LacZ. Figur 1 viser et rutediagram af den enzymatiske cellefusion assay. tTA transficeres ind v-celler, som udtrykker v-SNARE proteiner på celleoverfladen, og TRE-LacZ-transficeres ind i T-celler, der udtry...

Watch this Scientific Journal Video about Analysis of SNARE-mediated Membrane Fusion Using an Enzymatic Cell Fusion Assay at JoVE.com

Analysis of SNARE-mediated Membrane Fusion Using an Enzymatic Cell Fusion Assay

Vi har udviklet en cellefusion assay, der kvantificerer SNARE-medierede membranfusionsteknikker begivenheder ved aktiveret ekspression af β-galactosidase.

Analyse af SNARE-medieret membranfusion anvendelse af en enzymatisk Cellefusion Assay

The interactions of SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor)  proteins on vesicles (v-SNAREs) and on target membranes ...

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